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App. Sci. Report. 2 (2), 2013: 29-31 © PSCI Publications Applied Science Reports www.pscipub.com/ASR E-ISSN: 2310-9440 / P-ISSN: 2311-0139 Application of Recombinant DNA Technology- A review Shahin Gavanji Young Researchers Club, Khorasgan Branch, Islamic Azad University, Khorasgan, Isfahan, Iran. Corresponding author E-mail: [email protected] Key words Recombinant DNA Antibodies Drug delivery ABSTRACT Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure. They differ only in the nucleotides sequence within that identical overall structure. Pharmaceutical © 2013 PSCI Publisher All rights reserved. Introduction Biotechnology is the application of biological organisms, systems, or processes by various industries to learning about the science of life and the improvement of the value of materials and organisms such as pharmaceuticals, crops, and livestock. It is a relatively new and fast-developing field that integrates knowledge from several traditional sciences: biochemistry, chemistry, microbiology, and chemical engineering. The term itself is largely believed to have been coined in 1919 by Hungarian engineer Károly Ereky. In the late 20th and early 21st century, biotechnology has expanded to include new and diverse sciences such as genomics, recombinant gene technologies, applied immunology, and development of pharmaceutical therapies and diagnostic tests. Recombinant DNA molecules are sometimes called chimeric DNA, because they are usually made of material from two different species, like the mythical chimera. R-DNA technology uses palindromic sequences and leads to the production of sticky and blunt ends.The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA may be joined to bacterial DNA, or human DNA may be joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules. Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be created and introduced into any of a very wide range of living organisms. Proteins that result from the expression of recombinant DNA within living cells are termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein is not necessarily produced. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring of the foreign coding sequence. Recombinant DNA differs from genetic recombination in that the former results from artificial methods in the test tube, while the latter is a normal biological process that results in the remixing of existing DNA sequences in essentially all organisms(Miller and Nagarajan, 2000; Cederbaum et al., 1984). Recombinant DNA The idea of recombinant DNA was first proposed by Peter Lobban, a graduate student of Prof. Dale Kaiser in the Biochemistry Department at Stanford University Medical School (Lear, 1978) The first publications describing the successful production and intracellular replication of recombinant DNA appeared in 1972 and 1973(Jackson et al., 1973; Lobban and Kaiser, 1973). Stanford University applied for a US patent on recombinant DNA in 1974, listing the inventors as Stanley N. Cohen and Herbert W. Boyer; this patent was awarded in 1980 (Hughes, 2001). The first licensed drug generated using recombinant DNA technology was human insulin, developed by Genentech and Licensed by Eli Lilly and CompanyRecombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure. They differ only in the nucleotides sequence within that identical overall structure (figure 1). App. Sci. Report. 2 (2), 2013: 29-31 Figure 1. Construction of recombinant DNA, in which a foreign DNA fragment is inserted into a plasmid vector Application of Recombinant DNA Vectors in gene therapy A recombinant virus is a virus produced by recombining pieces of DNA using recombinant DNA technology. This may be used to produce viral vaccines or gene therapy vectors. Viruses All viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. Therefore this has been recognized as a plausible strategy for gene therapy, by removing the viral DNA and using the virus as a vehicle to deliver the therapeutic DNA. A number of viruses have been used for human gene therapy, including retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia, pox virus, and adeno-associated virus. Figure 2. Strategies for Delivering Therapeutic Transgenes into Patients(Thomas, 2006). 30 App. Sci. Report. 2 (2), 2013: 29-31 Recombinant Antibodies Developments in the fields of bacterial expression of functional antibodies and methods to select genes from a library by using the phenotype of the encoded polypeptide have been a breakthrough in antibody technology. Today, phage display in combination with antibody gene libraries is widely used to select E. coli host cells that express desired antibody fragments. Such gene libraries are typically produced either from natural sources (e.g., from the spleen of an immunized animal or from plasma cells of human donors) or generated by genetic engineering. The latter has been used to create naïve libraries based on one or more antibody VH and VL gene segments that are diversified by cassette mutagenesis or similar approaches. Such libraries are typically unbiased and can be used for any given antigen (Knappik et al., 2000; Soderlind et al., 2000; Hoet et al., 2005). Modern naïve libraries are generally large (more than 10 10 members), contain only few non-functional members, yield antibodies that are well expressed in E. coli (more than 1 mg of purified material per liter of culture) and are designed to allow further affinity maturation, if needed. Phage display is then most often used to select desired antibodies from such libraries(Hoet et al., 2005; Hoogenboom et al.,1998). Recombinant monoclonal antibodies The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected (Siegel, 2002). The phage antibody libraries are a variant of the phage antigen libraries first invented by George Pieczenik. These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications(Schmitz et al., 2000). Fermentation chambers have been used to produce these antibodies on a large scale. Conclusion Biotechnological drugs have the same high standard in quality and safety as conventional drugs. Of high interest is the question of costs of quality control for recombinant drugs. Integrating biotechnology and genomics in the whole drug development process gives companies the opportunity to save up to US$300 million per drug – about one-third of the costs today – and the prospect of bringing the drug two years earlier on the market Each day lost before market entry will lead to a loss of US$1.5 million per day, indicating the value of recombinant drugs and the need formakingmanufacturing processes operational and effective (Kayser and Müller, 2004). Any predictions for the near future are challenging. Future reports estimate a significant increase of recombinant drugs replacing up to 30% of commercially used low-molecular drugs up to 2015. For the production of recombinant biotech drugs, bioprocessing in all reactor sizes will be routinely used (Kayser and Müller, 2004). References Cederbaum SD, Fareed GC, Lovett MA, Shapiro LJ. 1984. Recombinant DNA in medicine. West J Med. 141(2):210-22. Griffiths AJF, Gelbart WM, Miller JH, Lewontin RC. 1999. Modern Genetic Analysis. 7rd ednNew York. Hughes S. 2001. Making dollars out of DNA The first major patent in biotechnology and the commercialization of molecular biology. Isis. 92 (3): 541–575. Hoet RM, Cohen EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, Rem L, Frans N, Daukandt M, Pieters H, van Hegelsom R, Neer NC, Nastri HG., Rondon IJ, Leeds JA, Hufton SE, Huang L, Kashin I, Devlin M, Kuang G, Steukers M, Viswanathan M, Nixon AE, Sexton DJ, Hoogenboom HR, Ladner RC. 2005. Generation of high-affinity human antibodies by combining donor-derived and synthetic complementaritydetermining- region diversity. Nat. Biotechnol. 23, 344–348. Hoogenboom HR, deBruine AP, Hufton SE, Hoet RM, Arends JW, and Roovers R C. ]1998. Antibody phage display technology and its applications. Immunotechnology. 4: 1–20. Jackson D, Symons R, Berg P. 1972. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: Circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc Natl Acad Sci 69 (10): 2904–2909. Kayser O, Müller RH. 2004. A Primer on Pharmaceutical Biotechnology and Industrial Applications. harmaceutical Biotechnology, Drug Discovery and Clinical Applications. 7rd edn. Wiley-VCH-Weinheim. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, Wolle J, Pluckthun A, Virnekas B. 2000. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296:57–86. Lobban P Kaiser A. 1973. Enzymatic end-to end joining of DNA molecules. J Mol Biol. 78 (3): 453–471. Miller JA, Nagarajan V. 2000. The impact of biotechnology on the chemical industry in the 21st century. Trends Biotechnol. 18: 109-191. Lear J. 1978. Recombinant DNA: 1rd edn The Untold Story. New York: Crown Publishers. Soderlind E, Strandberg L, Jirholt P, Kobayashi N, Alexeiva V, Aberg AM, Nilsson A, Jansson B, Ohlin M, Wingren C, Danielsson L, Carlsson R, Borrebaeck CA. 2000. Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat Biotechnol. 18: 852–856. Siegel DL .2002. Recombinant monoclonal antibody technology. Transfusion clinique et biologique : j Société franç trans san. 9 (1): 15–22. Schmitz U, Versmold A, Kaufmann P, Frank HG. 2000. Phage display a molecular tool for the generation of antibodies-a review. Placenta. 21:106–112. Thomas PZ. 2006. use of Genetically Modified Stem Cells in Experimental Gene Therapies. Regenerative Medicine. (1)1: 45-52. 31